Journal of Natural P&s
1430
Val. 58, No. 9,pp. 1430-1437, September 1995
ADDITIONAL BIOACTIVE ACETOGENINS, ANNOMUTACIN AND (2,d-TRAN.S AND CIS)-l OR-ANNONACIN-A-ONES, FROM THE LEAVES OF ANNONA MURlCATA FENGE W U , GENGXIANW O , LU ZENG, YAN Z ” G , JON T. SCHWEDLER, JERRY L. M&UGHLIN,* Department of Medicinal Chemistry and Pharmacognosy, School of Phaand Pharmacal Sciencu, Purdue University, West Lafayette, Indiana 47907 and SOELAKSONO
SASTRODIHARDJO
Department of Edwation and Culture, Center of Interuniversities, Biology Division, Bandung Institute of Technology, 10 Yanesba St., Bandung 40132, Indonesia ABSmn.-In a continuation of our research on bioactive components from the leaves of A n m &uta, three novel monotetrahydrofuranAnnonaceousacetogenins, namely,annomutacin 111,(2,4-trans)-10R-annonacin-A-one 121, and (2,4-ci()-lOR-annonacin-A-one131, have been identified. Their structures were deduced by ms, nmr, ir, and uv spectral and chemical methods, and the absolute configurations were determined by Mosher ester methodology. A known bioactive amide, N-p-coumaroyl tyramine, was also found. Compound 1 and the mixture of compounds 2 and 3 showed selective cytotoxicities against the human A-549 lung tumor cell line.
Annona muricata L. (Annonaceae)is a small tropical fruit tree whose seeds have yielded a number of cytotoxic and pesticidal monotetrahydrofuran (monoTHF) acetogenins (1-9). Previous studies with the leaves have resulted in the isolation of the novel mono-THF acetogenins, annomuricins A, B, and C (10,12),and muricatocins A, B, and C ( 1 1,12). In our continuing work, the leaves have now afforded the new bioactive mono-THF acetogenins,annomutacin El}
2
and a mixture of (2,4-trans)-1 OR-annonacin-A-one 121 and (2,4-cis)-10R-annonacin-A-one E37. The known bioactive amide,N-p-coumaroyl tyramine (13),was also isolated from the leaves. As described previously ( l o ) , the dried, powdered, leaves of A. muricata, obtained from plantation trees growing in Java, were extracted with 95% EtOH; the residue of the extract (F001) was partitioned through a standard extraction scheme (see Experimental), and the
trans
3 cis
September 19951
Wu et a/.: Acetogenins from Annona 468*558*+ -1 8 355337
7
.'
1431
141.
'' p 2 1 3 3 4 1 * * ~ 5 7 1 ~ 4 8 1 ~ 3 9 1 , ~ ? '
** ,,.+ 35
34/CH211g&(CH,l, 271* 6,
22
*8&
OR
0'18
:
17
OR
-18 241-2258
;
425%407389.'* 641%
385'90- 2 9 5 %
0 205
**
55 1 **
* R = H 111
** R=Me,Si
[lb]
FIGURE1. Diagnostic eims fragment ions (mlz)of annomutacin C11 and its pentaTMSi derivative [Ib]. -18 -18 (327) +309+291* 3 4 1 - % 2 5 1 v
471*38l%
291**
-90
OR
I
3 1 3 --223,' 90
* R=H 12 and 31 ** R=Me,Si E2b and 3bl
A 2
3
trans cis
FIGURE2. Diagnostic eims fragment ions (mlz) of 2 and 3 and their tri-TMSi derivatives [2b and 3bl.
partition residues were evaluated for toxicity with a test for lethality to brine shrimp larvae (BST) (14,15).The most active fraction (F005, BST LC,, 0.17 pg/ ml) was subjected to cc over Si gel eluted by gradients ofhexane/EtOAcand EtOAcI MeOH. The fractions were analyzed by tlc and evaluated in the BST assay (10).A pool of active fractions was further subjected to repeated cc and hplc to yield compound 1 and a mixture of 2 and 3. The known bioactive amide, N-pcoumaroyl tyramine, was isolated from another pool of active fractions using similar methods. Annomutacin 111and the mixture of (2,4-trans)-[21and(2,4-czs)-lOR-annonacin-A-ones 131 were obtained as colorless waxes. The ms (Figures 1 and 2 ) and nmr
spectra indicated that 1-3 are monoTHF ring acetogenins (4-6).The hrfabms gave EMHI' ions at mlz 625.5010 (calcd 625.5043) and 597.4706 (calcd 597.4730), consistent with molecular El1 and C,,H,O, formulas of C37H6807 E2 and 31, respectively. Compounds 1-3 showed a broad O H stretching absorption in the ir spectnun at 3250-3550 -1 cm . Four and three successive losses of H,O (mlz 18), respectively, from the [MH)' from 1 and the mixture of 2 and 3 in the cims, at mlz 607, 589, 571, and 5 5 3 117 and mlz 579,561,and 543 ( 2and 3), indicated the existence of four O H groups in 1and three O H groups in 2 and 3;these were confirmed by the formation of the tetra-acetate [la)and the tetratrimethylsilyl (TMSi) ether [lb] for 1
1432
Journal of Natural Products
Wol. 58,No. 9
eims spectra of the TMSi derivatives [lb and 2b, 3bl at mlz 641,571,385,271, and 2 13 for l b (Figure 1)and at mlz 541, 471,313,and 27 1 for 2b and 3b (Figure 2).The placement of the mono-THF ring 1 in 1 was determined to be at C-18/C-2 by the diagnostic fragments at mlz 641, 571,and 271,and, in 2 and 3, to be at C16/C-19by fragments at mlz 541, 471, and 271 (TMSi-eims). The it spectrum showed a strong absorption at 1765 cm-'
and the tri-acetates 12a and 3al and the tri-trimethylsilyl (TMSi) ethers f2band 3b] for 2 and 3. Compound l a gave four singlet proton peaks at 8 2.03,2.04, 2.05,and 2.08,and compounds 2a and 3a gave three singlet proton peaks at 8 2.04,2.05, and 2.08(Table 1).Thepositions of the O H groups in 1 were as-
signedatC-4,C-lO,C-l7,andC-22,and in 2 and 3 at C-10,C-15,and C-20,by careful analysis of the fragments in the
TABLE 1. 'H-Nmr Spectral Data for 1-3 and la-3a and "C-Nmr Data for 1 and the mixture of 2 and 3 (CDCI,, 6).
',cnmr
'H n m r (500 MHz) Positions
1
la
2q3a
1
.....
-
-
2
.....
-
3.09 m
3.09 m
2.40 m 2.52 m 3.84 rn
2.59 ddd 2.53 m 1.47 m 2.56 m 5.11 dq 4.55 E21 4.39 E31 dddd 1.27-1.62 m 1.27-1.57 4.87 m 3.59 m 1.41 m 1.57 m 1.27 m 1.27 m 1.27-1.62 m 1.27 m 3.40 m 1.27 m 3.81 m 1.57 m 4.87 dt 1.99 m, 1.63 m 1.92 m, 3.97 m 1.57 m 3.87 m 1.65-1.90 3.81 rn 1.65-1.90 1.37 m 3.97 m 1.27 m 4.87 dr 1.29-1.62 m 1.25-1.57 0.88 t 1.27 rn 3.04 dd 1.27 m 2.66 dd 0.88 t
2.59 dddd 1.47 rn 4.55 12a1 4.39 [3a)dddd 1.27-1.62 m 4.83 m 1.41 rn 1.27 m 1.27-1.62 m 4.83 m 3.96 rn 1.95, 1.72 rn
3a . . . . . 3b . . . .
4
.....
5-9
...
10 . . . . 11 . . . . 12 . . . . 13-14 . 15 . . . . 16 . . . . 17 . . . . 18
....
1.27-1.52 rn 3.60 m 1.37 rn 1.27 m 1.27 m 1.27 m 1.37 rn 3.42 rn 3.80 rn
19 . . . . 20 . . . . 21 . . . . 22 . . . . 23-31 . 32 . . . . 33a.. . . 33b . . . 34 . . . .
0.88 t
35 . . . . 36 . . . . 37 . . . . OAc-4. OAc-10 OAC-15 OAC-17 OAC-20 OAC-22
7.21 d 5.09 dq 1.41 d -
1.67-1.97 m 1.67-1.97 m 3.88 m 3.84 m 1.27-1.37 m 1.27 m 1.27 rn
2.17 s
-
1 174.40
7.09 d 5.02 dq 1.42 d 2.03 s 2.05 s 2.08 s 2.04 s
m
m m
rn
131.10 33.40 69.93 25-38 7 1.86 37.29 25.51 22-34 25.95 33.23 74.27
1.95, 1.72 m
82.15
3.96 m 4.91 m 1.37 m 1.27 m 1.29-1.62 m 0.88 t 3.04 dd 2.66 dd -
28.57 25.27 82.45 71.59 22-32 31.90 22.66
2.19 s -
151.78 77.94 19.10 -
-
2.05 2.08 2.04 -
s s s
14.09
178.66 127 178.68 131 44.25 121 44.79 131 25-37 79.30 [21 78.85 131 25-38 71.78 37.59 25.45 25-38 74.27 83.16 25-38
82.13 71.54 25-38 25-38 25-38 14.11 25-37 205.58 E21 205.78 13) 22.7 -
-
-
-
-
-
-
September 19951
W u et al.:
Acetogenins from Annonu
for the y-lactone and at 1715 cm-' for the ketone in 2 and 3. The mono-THF ring, with the usual O H groups on each side, was indicated, in 1 and the mixture of 2 and 3, by 'Hnmr chemical shifcs (Table 1)at 6 3.42 (H-17), 3.80 (H-18), 3.88 (H-21), and 3.84 (H-22), for 1, and 6 3.40 (H-15), 3.81 (H-16), 3.87 (H-19), and 3.81 (H20), for 2 and 3; and the l3C-nmr signals (Table 1) at 6 74.27 (C-17), 82.15 (C18), 82.45 (C-21), and 71.59 (C-22), for 1,and at 6 74.27 (C-15), 83.16 (C-16), 82.13 (C-l9),and71.54(C-20), for 2 and 3, confirmed this functionality. The carbinol proton at C-10 resonated at 6 3.60, for 1,and at 6 3.59, for 2 and 3, and the corresponding 13 C-nmr signal at 6 71.86, for 1,and at 6 71.78, for 2 and 3. The nmr chemical shifts of H-4 and C-4 in 1 were located at 6 3.84 and 69.93 (Table l ) , respectively. These structural units were further confirmed by COSY and single-relayed COSY data in which the proton coupling correlations from H-3"-4, and H10"-8, H-9 and H - l l ) , and (H-17 and H - 2 2 ) ~ ( H - 1 5H-16, , H-18, H-19, and H-20, H-21, H-23, and H-24), in 1, and H-10"-8, H-9, H-11), and (H15 and H-20)"-13, H-14, H-16, H17, and H-18, H-19, H-21, and H-22), in 2 and 3 (Table 1)could be clearly seen. The assignments of the relative stereochemistry around the mono-THF ring of 1, la, 2,2a,and 3,3a were determined using the methodology of Hoye and coworkers (16,17) and Born et al. (18), as well as by comparison with several acetogenins having the erythro configuration at C-19 to C-20, for example, annonacin A (19), (2,4-cis and trans)annonacin-A-one (20), and jetein (2 1).In addition, the recent paper by Fujimoto et al. (22) describes model mono-THF analogues, with flanking hydroxyls, having all possible relative stereochemistries;and 1 and the mixture of 2 and 3 match with the threo-trans-erythro model. The OHsubstituted CH centers, at C-17 and C-
1433
22 in 1,and at C-15 and C-20 in 2 and 3, flanking the ring region (C-18 to C-21 and C-16 to C- 19),exhibited very similar chemical shifts in the 'H- and 13C-nmr spectra as with the above models (Table 1).The proton signals for H-17 at 6 3.42 and H-22 at 6 3.84, in 1,and for H-15 at 6 3.39 and H-20 at 6 3.87, in 2 and 3, were shifted downfield in l a to 6 4.87 for H-17 and 6 4.87 for H-22, and in 2aand 3a to 6 4.83 for H-15 and 6 4.89 for H20. The stereochemistry of C-17/C-18 and C-21K-22 in 1, and C-15/C-16 and C-19/C-20 in 2 and 3, was thus concluded to be threo and erythro for 1-3, and the stereochemistry is trans for the THF ring in each compound (16,17). Annomutacin 111 is analogous to annomontacin (23) which is threo-transthreo at C-17 to C-22. The OH-substituted CH center at C-22 is the only difference between these two compounds. The carbinol protons at C-22 in annomontacin and annomutacin {l]resonated at 6 3.42 and 3.84, and the corresponding 13C-nmrsignals were at 6 74.32 and 7 1.59,respectively. Thus, the stereochemistry of C-21/C-22 in annomutacin E11 was concluded to be erythro. The other spectral data of annomontacin (23) and annomutacin 111 are identical. The 'H- and 13C-nmrspectral dataof the mixture of (2,4-trans and cis)-1ORannonacin-A-one {2 and 31 were essentially identical with those of the mixture of (2,4-cis and trans)-annonacin-A-one isolated from Asimina triloba (20), and the shape of the 'H-nmr signal at 6 3.40 (C10) of both of these two pairs of compounds was their only difference. The peak at 6 3.40 was split as a pseudoquartet in the mixture of 2 and 3, but appeared as a multiplet in (2,4-cis and trans)-annonacin-A-one. The differences between these compounds were, thus, believed to be due to different absolute configurations of the carbinol centers at C-10. The absolute stereochemistry at C10 of (2,4-cis and trans)-annonacin-Aone has not been reported (20), although
1434
Wol. 58, No. 9
Journal of Natural Products
the absolute stereochemistry of isoannonacin is C-1OR (25). Examination in the 'H-nmr spectrum of the H-10 resonance of isoannonacin showed a signal identical to those of H-10 in the mixture of 2 and 3 and supported our suspicion that 2 and 3 must be C-1OR. Rieser et al. have reported the determination of the absolute configuration of stereogenic carbinol centers in several Annonaceous acetogeninsusing advanced Mosher ester methodology (6,24). Thus, the per-(S)- and (R)-methoxytrifluoromethyl phenylacetic acid (MTPA) esters (Mosher esters) of 1 and the mixture of 2 and 3 were prepared and numbered ls,lr; 2s,2r; and 3s,3r, respectively. COSY
'H-nmr analysis of these derivatives was then performed. The 'H-nmr chemical shift data of 1s and l r showed that the absolute configuration at C-4 of 1 is R (Table 2). This result is identical to all acetogenins examined so far that possess an OH group at C-4. The Mosher ester data also suggested the absolute stereochemical assignments of the carbinol centers adjacent to the mono-THF ring as C-17R and C-22S, in 1 (Table 3), and as C-15R and C-20S, in 2 and 3 (Table 4). The determination of the absolute stereochemistry of the carbinol center at C-10 of 1 could not be achieved by direct application of the Mosher-ester method because the 'H-
TABLE2. 'H-Nmr Chemical Shifts for the Determination of the Absolute Confieuration at C-4 of the Tetra (S)-and (R)-MTPA Esters of 1. MTPA ester of
CH,-5
H-4
l r 8 ( R ) .. . . . . . . A S . . ..........
1.57 1.56 +0.02
5.30 5.36 -0.06
2.57 2.65 -0.08
MTPA Ester of
CH,-16
H-17
1s . . . . . . . . . . . . 8(S)... . . . . . . . . lr . . . . . . . . . . . . 8(R)
..........
1.55 1.48 1.50
1.44
A8 . . . . . . . . . . . .
pos.
ls8(S) . . . . . . . .
TABLE4.
CHZ-3
H-33
H-34
Me-35
2.52 2.56 -0.04
6.72 6.95 -0.23
4.85 4.89 -0.04
1.27 1.29 -0.02
H-18
19/20 CH2-
H-21
H-22
CH,-23
5.21
3.93
3.92
5.01
5.26
3.98
1.86 1.65 1.83 1.57
3.74
4.96
neg.
neg.
pos.
pos.
pos.
1.53 1.45 1.56 1.49 neg.
Configuration
4R
configuration
17R, 22s
'H-Nmr Chemical Shifts for the Determination of the Absolute Configurations at C-15 and C-20 of the Tri 6)and (R)-MTPA Esters of 2 and 3.
MTPA Ester of
2s,3s 8 (S) . . . . . . . 2r,3r8 (R) . . . . . . . A8............
CH,-14
H-15
H-16
17/18 CH2-
H-19
H-20
1.53 1.46 1.48 1.42
5.21
3.90
3.91
5.06
5.26
3.97
3.73
5.01
pos.
neg.
neg.
1.86 1.65 1.83 1.57 pos.
pos.
pos.
CH,-21
Configuration
1.51 1.43 1.54 1.47 neg. 15R, 20s
September 19951
W u et al.: Acetogenins from Annona
nmr chemical shifts at CH,-8, CH,-9, CH,- 11, and CH,- 12 in the MTPA esters (ls,lr) cannot be confidently assigned. However, in the mixture of 2 and 3, the 1 H-nmr chemical shifts at CH-4 are affected by the MTPA ester at C-10 and can be assigned for each isomer (Table 5). Thus, the absolute configuration of the carbinol center at C-10 was easily deduced as 10R for 2 and 3 by analysis of the Mosher ester data. Consequently, 2 and 3 are, indeed, the ketolactones of an annonacin A-type of mono-THF acetogenin (26,27). We have named 2 and 3, respectively, (2,4-transand cis)-1OR-annonacinA-one; and we propose that the (2,4-cis and trans)-annonacin-A-ones previously reported (20) from Asimina triloba are C10s. Annomutacin 111and the mixture of (2,4-trans and cis)-10R-annonacin-A-ones 12 and 31 were significantly bioactive in the BST; they were selectively cytotoxic (seven-day MTT assays) to the human A549 solid lung tumor cell line with 2 and 3 showing moderate activity against the breast cell line (MCF-7) (Table 6). All of the Annonaceous acetogenins act, at
MTPA Ester of
2s 6 (S) . . . . . . . . 21 6 (R) . . . . . . . .
A6 . . . . . . . . . . . .
I
H-4
I
4.53 4.51 0.02
TABLE6.
1' . . . . . . . . . . . . .
2,3'.. . . . . . . . . . Adriamycin'" . . . .
I
I
1
1435
least in part, as potent inhibitors of complex I in mitochondria (28,29). EXPERIMENTAL GENERAL EXPERIMENTAL described previously (10).
PROCEDURES.-AS
F"TMATERIAL.-ASdescribed previously (10).
EXTRACTIONAND IsouTIoN.-For the column chromatographic separation of F005, a total of 154 fractions was collected. The BST-active fractionsNos. 90-95 andNos. 76-85 were further subjected to repeated flash chromatography to yield crude compounds of 1and the mixture of 2 and 3,respectively; each was then purified by hplc over Si gel, eluted with hexane-MeOH (90:1, flow rate 10 mlimin), to afford, as colorless waxes, 1and a mixture of 2 and 3. A bioactive amide was isolated from other fractions (Nos. 86-87), using similar methods, and was identified as N-pcoumaroyl tyramine (13). Annomtrtucin [l].-Colorless wax (7 mg); [ a ] * ' f60.0" ~ ( ~ 0 . 0 0 1EtOH); , hifabms (glycerol) mlz [MH]+ 625.5010 for C37H6907 (calcd
625.5043); cims (n-BuOH) mlz 625 (loo), 607 (46), 589 (23), 571 (l), 553 (I), 425 (I), 407 (I), 389(1), 355(1),337(1),241(1),223(1), 199(1), 141 (1); 'H-nmr (CDCI,, 500 MHz) data, see Table 1; 13C-nmr (CDCI,, 125 MHz) data, see Table 1; ir (film) v max 3387 (br OH), 292 1,285 1,
1743,1466,1320,1073cm-';uvXmax(MeOH) 212 nm (e=9.2X103).
MTPAEster of
3s 6 (S) 31 6 (R) A8
I
1
Configuration
H-4 4.37 4.35 0.02
I
4R, 10s
Bioactivities of 1and the Mixture of 2 and 3.
3.91XlO-* 1.12x lo-' 3.13X10-'
I
1.57X10-' 1.74X10-' 3.56X lo-'
I
>1.0 5.70X lo-' 1.42X10-'
I
>1.0 >1.0 3.01 X lo-*
'Brine shrimp lethality test (14,15). bHuman lung carcinoma (30). 'Human breast carcinoma (31). dHuman colon adenocarcinoma (32). 'Same cytotoxicity runs; values in different runs were within one order of magnitude of each other. 'Positive control standard.
1436
Journal of Natural Products
Tetra-acetate [la].- Eims mlz 55 1 (5), 491 (9), 481 (2), 431 (11), 421 (I), 361 (2), 325 (3), 141 (6); ’H-nmr (CDCI,, 500 MHz) data, see Table 1. (2,4-Trans andcis)-1 OR-annomin-A-ones [Z a d 3].-As a colorless wax, a mixture of both 2 and 3 (7 mg); [a]”D +15.0° ( ~ 0 . 0 0 2 )hrfabms ; (glycerol)m/z[MI-E? 597.4706forC,,H6,0, (calcd 597.4730); cims m/z [MH]’ 597 ( 4 4 , 579 (43), 561 (loo), 543 (25), 395 (2), 385 (I), 379(3), 367 (21,361 (3), 309(6), 291 (4), 241 (9), 223 (2), 141 (2); eims m/z 395 (l),385 (2), 379(2), 367 (3), 361 (3), 327 (2), 309(7), 291 (5), 241 ( 3 , 2 2 3 (3), 141 (5); ’H-nmr (CDCI,, 500 MHz) data, see Table 1; 13 C-nmr (CDCI,, 125 MHz) data, see Table 1; ir (film) u max 3443 (br OH), 2920, 5851, 1765, 1715, 1465, 1356, 1166, 1075 cm- ; uv A max (MeOH) 208 nm (E 2.5X103). Tri-acetates [2a,3a].- ’H-Nmr(CDCI,, 500 MHz) data, see Table 1. TMSi derivatives [lb,2b, and 3b].-Eims of lbm/z714(10),641(16), 571 (100),558(1),551 (36), 481 (63), 461 ( l l ) , 385 (95), 356 (36), 341 (391,295 (18), 271 (77), 213 (46), 205 (12), 169 (36); eimsof2band 3 b m h 812 (2), 601 (lo), 541 (16),511(10),499(2),471(59),409(3),381(21),
361 (9), 341 (20), 319(3), 313 (41), 291 (7), 271 ( 4 9 , 2 5 1 (3), 213 (4), 181 (2), 141 (6). ACKNOWLEDGMENTS This work was supported, in part, by R01 grant No. CA 30909 from the National Cancer Institute, NIH. One of the authors, F.-E. Wu, Chengdu Institute of Biology, the Chinese Academyofkiences, People’sRepublic ofChina, gratefully acknowledges the support of the K.C. Wang Education Foundation, Hong Kong. The plant material was collected with travel support from the Indonesian University Development Projects, which were financed by the World Bank. LITERATURE CITED 1.
2.
3.
4.
5. 6.
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Received 12 October 1994
